Ex vivo method for determining potential GLP-2 receptor modulators

ABSTRACT

Disclosed herein is a method for measuring the contractility of intestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Also disclosed is an assay which directly measures the activity of GLP-2 or GLP-2 ligands ex vivo and permits the screening of putative GLP-2 ligands in native tissue.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 60/741,075 filed on Dec. 1, 2005, the entire contents of which are hereby incorporated by reference. This application claims priority under 35 U.S.C. 120 to U.S. Non-provisional application Ser. No. 11/607,030, now U.S. Pat. No. 7,498,141.

BACKGROUND OF THE INVENTION

Glucagon-like peptide-2 (GLP-2) is a 33-amino acid proglucagon-derived peptide secreted by the endocrine L-cell primarily in the lower gastrointestinal tract in response to luminal nutrients. Plasma levels have been shown to significantly increase within an hour of ingesting a meal, in particular following the ingestion of carbohydrates or fat (1). GLP-2 has been shown to be responsible for the regulation of proliferation and apoptosis of the intestinal epithelium (2,3). These changes in part result in an increase in mucosal surface area, enhanced absorptive efficiency and barrier function in the small intestine (4-6). GLP-2 also decreases gastric motility, inhibits gastric acid secretion, increases nutrient transport activity and acutely increases intestinal and portal blood flow (7-11).

GLP-2 and GLP-2 analogues promote the growth and repair of the intestinal epithelium in models of disease, including enhanced adaptation and nutrient absorption following small bowel resection and alleviation of TPN-induced hypoplasia in rodents (12-15). GLP-2 analogues have demonstrated decreased mortality and improvement of disease-related histopathology in animal models of intestinal damage such as indomethacin-induced enteritis, dextran sulfate-induced colitis and chemotherapy-induced mucositis (16-19).

The intestinotrophic effects of GLP-2 are mediated by the GLP-2 receptor (GLP-2R), a member of the superfamily of G-protein coupled receptors and most closely related to the GLP-1 and glucagon receptor gene subfamily (20). The GLP-2R is a high-affinity, ligand-specific functional receptor coupled to the G-protein Gs. Studies of the activation of the cloned GLP-2R by GLP-2 analogues show a correlation of in vitro activity with in vivo intestinotrophic efficacy (20).

To date, characterization of GLP-2R function has been limited to heterologous cell line expression, mucosal fractions and primary cell cultures. GLP-2 selectively stimulates cAMP production in recombinantly expressing GLP-2R cell lines, isolated intestinal mucosal fractions containing enteroendocrine and neural cells and primary hippocampal cultures (21-24). The study of native GLP-2R biological activity has focused on in vivo animal models and ex vivo model systems that require exogenous application of GLP-2 to the whole animal prior to tissue isolation (2-19, 25-31). There is no ex vivo method for characterizing putative GLP-2 ligands directly; such a method would have obvious utility, for example in the screening and characterization of GLP-2 ligands of pharmaceutical interest.

SUMMARY OF THE INVENTION

Disclosed herein is a method for measuring the contractility of intestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Thus, one aspect of the invention is an assay which directly measures the activity of GLP-2 or GLP-2 ligands. Another aspect of the invention is the direct characterization of the GLP-2 receptor. A further aspect of the invention is a method for screening compounds for activity at the GLP-2 receptor, such as putative GLP-2 agonists, antagonists, modulators or the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the organ bath trace of the effect of 1 uM GLP-2(1-33) on rat contractility in duodenum, jejunum, ileurn and colon.

FIG. 2 shows the organ bath trace of the effect of GLP-2(1-33) on rat colon contractility.

FIG. 3 shows the organ bath trace of the effect of [gly2]GLP-2 on rat colon contractility. Teduplutide inhibited spontaneous colon contractility in a concentration-dependent manner.

FIG. 4 shows the inhibitory effects of GLP-2 analogues on spontaneous contractility in rat colon.

FIG. 5 shows the effect of GLP-2 analogues on cAMP accumulation in EBNA293 cells stably expressing rat GLP-2R.

FIG. 6 shows the effect of GLP-2(3-33) on GLP-2(1-33)-induced inhibition of colon contractility. GLP-2(1-33) inhibited contractility with an estimated IC₅₀ of 13.5±5.4 nM (n=9). GLP-2(3-33) blocked GLP-2(1-33)-induced inhibition of colon contractility. The GLP-2(1-33) concentration response curves shifted to the right with estimated IC₅₀ of 95.5±32.5 nM (n=5) and 142.7±55.9 nM (n=5) at concentrations of 1 μM and 10 μM respectively.

FIG. 7 shows the effect of GLP-2(3-33) on teduglutide-induced inhibition of rat colon contractility. Teduglutide inhibited contractility with an estimated IC₅₀ of 4.9±1.9 nM (n=6). GLP-2(3-33) blocked teduglutide-induced inhibition of colon contractility The teduglutide concentration response curves shifted to the right with estimated IC₅₀ of 34.7±15.1 nM (n=4) and 59.9±11.9 nM (n=5) at 1 μM and 10 μM GLP-2(3-33) respectively.

FIG. 8 shows the effect of a small molecule antagonist on GLP-2(1-33)-induced inhibition of colon contractility. GLP-2(1-33) concentration response curves shifted to the right in the presence of 1 μM and 10 μM antagonist respectively.

FIG. 9 shows the effect of a small molecule positive modulator on GLP-2(1-33)-induced cAMP accumulation. GLP-2(1-33) concentration response curves shifted to the left and increased maximal stimulation in the presence of 1 μM and 10 μM modulator, respectively.

FIG. 10 shows the effect of a small molecule positive modulator on GLP-2(1-33)-induced inhibition of colon contractility. GLP-2(1-33) concentration response curves shifted to the left and downward in the presence of 1μ and 10 μM modulator respectively.

DETAILED DESCRIPTION OF THE INVENTION Definitions

hGLP-2R is the human GLP-2 receptor; rGLP-2R is the rat GLP-2 receptor. hGLP-2 is human GLP-2 (also known as Teduglutide); rGLP-2 is rat GLP-2.

A “GLP-2 ligand” is defined as any molecule which interacts with the GLP-2 receptor; such molecules may be peptides (such as analogues of naturally occurring GLP-2) or small molecules.

A “modulator” is defined as a molecule which modulates the activity of a GLP-2 agonist.

“Intestinal tissue” is broadly defined as intestine from the pylorus to the rectum. “Small intestine” is defined as intestine from the pylorus to the ileo-cecal valve (it may also be defined as duodenum, jejunum and ileum). “Large intestine” is defined as intestine distal from the cecum (it may also be defined as colon tissue).

Disclosed herein is an assay which, for the first time, demonstrates the ability of GLP-2 receptor ligands to directly affect native GLP-2R-mediated effects in colon tissue ex vivo. As a result, it is possible to directly screen and pharmacologically characterize GLP-2 ligands, such as peptidic analogies of GLP-2, as well as small molecule agonists, antagonists and modulators, against whole tissue.

Such tissue is of mammalian origin; in one aspect it is of rat origin, in others it is of mouse or guinea-pig origin.

The assay method comprises measuring the contractility of segments of intestinal tissue ex vivo. The fact that this assay is useful for the stated purpose is not obvious, based upon the known expression levels of the GLP-2 receptor in this tissue. Further, previous studies have shown that direct application of GLP-2 ex vivo to this tissue has no effect.

One aspect of the invention is a method for characterizing the GLP-2 receptor; for example in an endeavour to better understand the physiological role of GLP-2 and the GLP-2 receptor in intestinal tissue or to elucidate the role of putative mediators related to GLP-2 activation and physiology.

Another aspect of the invention is a method for determining whether a compound is active on the GLP-2 receptor comprising the step of measuring contractility of intestinal tissue segments ex vivo in the presence and absence of compound, wherein a difference in contractility in the presence of the compound is indicative of activity on the GLP-2 receptor.

Yet another aspect of the invention is a method for determining whether a compound acts as a GLP-2 receptor agonist comprising the step of measuring contractility of intestinal tissue segments ex vivo in the presence and absence of compound, wherein inhibition of contractility in the presence of the compound is indicative of GLP-2 receptor agonist activity.

Yet another aspect of the invention is a method for determining whether a compound acts as a GLP-2 receptor antagonist comprising the step of measuring the effect of a GLP-2 receptor agonist on contractility of intestinal tissue segments ex vivo in the presence and absence of the compound, wherein a decrease of agonist inhibition of contractility in the presence of the compound is indicative of GLP-2 receptor antagonist activity.

Another aspect of the invention is a method of assaying compounds for activity at the GLP-2 receptor comprising the steps of:

i. Obtaining a segment of intestinal tissue,

ii. Suspending the segment in an organ bath,

iii. Placing the segment under tension,

iv. Incubating the segment with a compound of interest and

v. Measuring the contractility of the tissue; wherein an effect on contractility of the segment is indicative of activity on the GLP-2 receptor.

In one aspect the tissue is intestinal tissue. In another it is small intestine tissue. In yet another it is large intestine tissue or colon tissue. FIG. 1 shows the Organ Bath Trace of the effect of 1 uM GLP-2(1-33) on rat contractility in duodenum, jejunum, ileum and colon. FIG. 2 shows that GLP-2(1-33) inhibits spontaneous colon contractility in a concentration-dependent manner in rat colon tissue; FIG. 3 shows the analogous effect of Teduglutide.

The assay of putative agonists may be conducted by incubation of the colon segments with a predetermined concentration of the ligand to be tested for 5 to 10 minutes. FIG. 4 shows typical results for a number of GLP-2 analogues. FIG. 5 shows the effect of GLP-2 analogues on cAMP accumulation in EBNA293 cells stably expressing rGLP-2R.

The assay of putative antagonists may be conducted by pre-incubation of the colon segments with a predetermined concentration for, for example, 5 to 10 minutes of the ligand to be tested followed by addition and incubation of a predetermined concentration of a GLP-2 agonist. FIGS. 6, 7 and 8 show typical results for a number of GLP-2 antagonists; concentration-response curves are clearly right-shifted, as expected.

The assay of putative modulators, may be conducted by pre-incubation of the colon segments with a predetermined concentration of the ligand to be tested for, for example, 5 to 10 minutes followed by the addition and incubation of a pre-determined concentration of a GLP-2 agonist. FIG. 10 show typical results for a GLP-2 positive modulator; the effect of the modulation is clearly demonstrated. FIG. 9 shows the corresponding effect on GLP-2(1-33)-induced cAMP accumulation.

Methods & Results

It should be noted that the methods described below are illustrative in nature, and that the invention is not limited to the particular embodiments described.

Segments of colon from male Sprague Dawley rats were suspended in organ bath chambers containing Krebs' solution and maintained at 37° C. with 95% O₂ and 5% CO₂. Basal tone, contractility rate and peak height were used to measure the properties of spontaneous and ligand-mediated colonic contractions. Segments were initially loaded to a tension of 2 g and allowed to equilibrate for 60 minutes. During this period, segments were repeatedly washed every 10 minutes. [gly2]GLP-2 (“Teduglutide”, a GLP-2 agonist) and GLP-2 analogues were subsequently incubated with each tissue segment for 5 to 10 minutes. The tissues were allowed to re-equilibrate with repeated washes following each experiment.

Teduglutide and GLP-2(1-33) inhibited muscle contractility in a concentration-dependent manner with an estimated IC₅₀ of 4.9±1.9 (SE) nM (n=6) and 13.5±5.4 nM (n=9), respectively. While having no effect alone, GLP-2(3-33) (a GLP-2 antagonist/partial agonist) blocked the inhibitory effects of Teduglutide and GLP-2. In the presence of 10 μM GLP-2(3-33), Teduglutide and GLP-2(1-33) inhibited colon contractility with an IC₅₀ of 59.9±11.9 nM (n=6) and 142.7±55.9 nM (n=5) respectively. The rank orders of potency of GLP-2 peptide analogues were similar to that previously reported for their cAMP activation in cell lines recombinantly expressing the rGLP-2R.

These studies demonstrate the ability of GLP-2 to reproducibly and significantly reduce spontaneous contractility of rat colon segments suspended in organ bath chambers. GLP-2-induced inhibition of colon contractility is concentration-dependent and reversible following a wash-out period. The GLP-2R antagonist/partial agonist GLP-2(3-33) blocks the effect of GLP-2(1-33) in a concentration-dependent manner. The rank order of potency of GLP-2 analogues on the inhibition of colon contractility was comparable to the rank order of increased cAMP accumulation in heterologous cells expressing GLP-2R.

Therefore the described assay enables native GLP-2R characterization by the direct application of GLP-2 to intestinal tissue segments ex vivo. As a result, it is possible to screen and pharmacologically characterize GLP-2 peptide analogues, GLP-2R small molecule agonist and antagonists against whole tissue.

Colon Contractility—Methods

Animals

Male Sprague-Dawley (SD) rats (Charles River Canada, Quebec), weighing 200-250 g, were used for these studies. The animals were housed on contact bedding and kept with free access to food (Purina Rodent Diet) and water in a facility with regulated temperature and humidity, under an alternate 12-hour light/dark cycle. Animals were acclimatized for 3˜5 days prior to harvesting of tissues.

Tissue Preparation

Rats were euthanized by decapitation. The entire intestine was quickly removed and placed in a Petri dish containing a Sylgard layer and 37° C. Krebs' solution bubbled with 95% O₂ and 5% CO₂. The Krebs' solution (pH 7.4) contained 118.1 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄.7H₂O, 2.5 mM CaCl₂.2H₂O and 11 mM glucose. The colon was isolated, cleaned of superficial fat and mesentery and sectioned into eight 1-cm long segments. The lumen was cleaned by carefully flushing each segment with Krebs' solution using plastic transfer pipets. Segments were transferred to a separate Petri dish containing fresh Krebs' solution and suture threads were tied to both ends of each segment. Tissues were suspended longitudinally using surgical suture, one end attached to the transducer on the top and the other to the tissue holder at the bottom of 20 ml organ bath chambers containing Krebs' solution, maintained at 37° C. with 95% O₂ and 5% CO₂. Isometric tensions were continuously measured using calibrated force displacement transducers and data was collected and processed by the MP100WSW system with AcqKnowledge software

Experiment Protocol

Segments of colon were initially loaded to a tension of 2 g and allowed to equilibrate for a 60 min period. During this period, segments were washed with Krebs' solution at 10 minutes intervals. When the tissues obtained regular and stable contraction patterns, GLP-2 agonist response experiments were conducted by the addition of eight concentrations of compound into eight separate organ baths for 5-10 minutes. Routinely, one-hour washes were interspersed between repeat applications of compounds to allow for recovery. The effect of GLP-2(3-33) on GLP-2 agonist analogues were conducted by pre-incubating colon segments with 1 or 10 uM GLP-2(3-33) for 5-10 minutes. Then agonist responses were measured at various concentrations as described above.

Data Analysis

Tension was measured by changes in peak height. Data were calculated and expressed as percentage response relative to the basal level (prior to the experiment). Graphs and IC₅₀ determinations were plotted by the percentage response in GraphPad Prism (v 3.02). In order to assess the significance of the effect of GLP-2 and Teduglutide paired t-tests were conducted on the Log [IC₅₀] values.

REFERENCES

-   1. Xiao Q., Boushey R. P., Drucker D. J., Brubaker P. L. Secretion     of the intestinotrophic hormone glucagon-like peptide 2 is     differentially regulated by nutrients in humans. Gastroenterol.     1999; 117:99-105 -   2. Drucker D. J., Ehrlich P., Asa S. L., Brubaker P. L. Induction of     intestinal epithelial proliferation by glucagon-like peptide-2.     Proc. Natl. Acad. Sci. USA 1996; 93:7911-16 -   3. Tsai C.-H., Hill M., Drucker D. J. Biological determinants of     intestinotrophic properties of GLP-2 in vivo. Am. J. Physiol. 1997;     272:828-32 -   4. Tsai C. H., Hill M., Asa S. L., Brubaker P. L., Drucker D. J.     Intestinal growth-promoting properties ofglucagon-like peptide-2 in     mice. Am. J. Physiol. 1997; 273:E77-84 -   5. Brubaker P. L., Izzo A., Hill M., Drucker D. J. Intestinal     function in mice with small bowel growth induced by glucagon-like     peptide-2. Am. J. Physiol. 1997; 272:E1050-8 -   6. Benjamin M. A., McKay D. M., Yang P.-C., Cameron H., Perdue, M H.     Glucagon-like peptide-2 enhances intestinal epithelial barrier     function of both transcellular and paracellular pathways in the     mouse. Gut 2000; 47:112-9 -   7. Wojdemann M., Wettergren A., Hartmann B., Holst J. J.     Glucagon-like peptide-2 inhibits centrally induced antral motility     in pigs. Scand. J. Gastroenterol. 1998; 33:828-32 -   8. Wojdemann M., Wettergren A., Hartmann B., Hilsted L., Holst J. J.     Inhibition of sham feeding-stimulated human gastric acid secretion     by glucagon-like peptide-2. J. Clin. Endocrinol. Metab. 1999;     84:2513-17 -   9. Guan X., Stoll B., Lu X., Tappenden K. A., Holst J. J., Hartmann     B., Burrin D. GLP-2-Mediated Up-Regulation of Intestinal Blood Flow     and Glucose Uptake Is Nitric Oxide-Dependent in TPN-Fed Piglets.     Gastroenterol. 2003; 125: 136-147 -   10. Cheeseman C. I., Tsang R. The effect of gastric inhibitory     polypeptide and glucagon like peptides on intestinal hexose     transport. Am. J. Physiol. 1996; 271:G477-G482 -   11. Cheeseman, C. I. Upregulation of SGLT-1 transport activity in     rat jejunum induced by GLP-2 infusion in vivo. Am. J. Physiol. 1997;     273:R1965—R1971 -   12. Scott R. B., Kirk D., MacNaughton W. K., Medding J. B. GLP-2     augments the adaptive response to massive intestinal resection in     rat. Am. J. Physiol. 1998; 38:G911-21 -   13. Sigalet D. L., Martin G. R. Hormonal therapy for short bowel     syndrome. J. Pediatr. Surg. 2000; 35:360-4 -   14. Chance W. T., Foley-Nelson T., Thomas I., Balasubramaniam A.     Prevention of parenteral nutrition-induced gut hypoplasia by     coinfusion of glucagon-like peptide-2. 1997; 273:G559-63 -   15. Chance W. T., Sheriff S., Foley-Nelson T., Thomas I.,     Balasubramaniam, A. Maintaining gut integrity during parenteral     nutrition of tumor-bearing rats: effects of glucagon-like peptide 2.     Nutrition and Cancer 2000; 37:215-22 -   16. Boushey R., Yusta B., Drucker D. J. GLP-2 decreases mortality     and reduces the severity of indomethacin-induced murine enteritis.     Am. J. Physiol. 1999; 277:E93747 -   17. Drucker D. J., Yusta B., Boushey R. P., DeForest L.,     Brubaker P. L. Human [Gly2]GLP-2 reduces the severity of colonic     injury in a murine model of experimental colitis. Am. J. Physiol.     1999; 276:G79-91 -   18. Boushey R. P., Yusta B., Drucker D. J. Olucagon-like peptide     (GLP)-2 reduces chemotherapy-associated mortality and enhances     survival in cells expressing a transfected GLP-2 receptor. Cancer     Res. 2001; 61:687-93 -   19. Tavakkolizadeh A., Shen R., Abraham P., Kormi N., Seifert P.,     Edelman E. R., et al. Glucagon-like peptide 2: A new treatment for     chemotherapy-induced enteritis. J. Surg. Res. 2000; 91:77-82 -   20. Monroe D. G., Gupta A. K., Kooshesh P., Vyas T. B., Rizkalla G.,     Wang H., et al. Prototypic G protein-coupled receptor for the     intestinotrophic factor glucagon-like peptide-2. Proc. Natl. Acad.     Sci. USA 1999; 96:1569-73 -   21. Yusta B. Somwar R., Wang F., Munroe D., Grinstein S., Klip A.,     et al. Identification of glucagon-like peptide-2 (GLP-2)-activated     signaling pathways in baby hamster kidney fibroblasts expressing the     rat GLP-2 receptor. J. Biol. Chem. 1999; 274:30459-67 -   22. Yusta B., Boushey R. P., Drucker D. J. The glucagon-like     peptide-2 receptor mediates direct inhibition of cellular apoptosis     via a cAMP-dependent protein kinase-independent pathway. J. Biol.     Chem. 2000; 275: 35345-52 -   23. Walsh, N. A., Yusta, B., DaCambra, M. P., Anini, Y., Drucker, D.     J., Brubaker, P. L. Glucagon-like peptide-2 receptor activation in     the rat intestinal mucosa. Endocrinology 2003; 144(10):4385-92 -   24. Lovshin, J. A., Huang, Q., Seaberg, R., Brubaker, P. L.,     Drucker, D. J. Extrahypothalamic Expression of the Glucagon-like     Peptide-2 (GLP-2) Receptor is Coupled to Reduction of     Glutamate-induced Cell Death in Cultured Hippocampal Cells.     Endocrinology 2004; 145: 3495-506 -   25. Burrin D. G., Stoll B., Jiang R., Petersen Y., Elnif J.,     Buddington R. K. et al. GLP-2 stimulates intestinal growth in     premature TPN-fed pigs by suppressing proteolysis and apoptosis.     Am. J. Physiol. 2000; 279:G1249-56 -   26. Kouris G. J., Rossi Q. L., Djuricin G., Nathan G. C.     Weinstein R. A., Prinz R. A. The Effect of Glucagon-like Peptide 2     on Intestinal Permeability and Bacterial Translocation in Acute     Necrotizing Pancreatitis. Am. J. Surg. 2001; 181:571-575 -   27. Prasad R., Alavi K., Schwartz M Z. Glucagon-like peptide-2     analogue enhances intestinal mucosal mass after ischemia and     reperfusion. J. Pediatr. Surg. 2000; 35:357-9 -   28. Prasad R., Alavi K., Schwartz M Z. GLP-2□ accelerates recovery     of mucosal absorptive function after ischemia/reperfusion. J.     Pediatr. Surg. 2001; 36: 570-572 -   29. Alavi K., Schwartz Z., Palazzo J. P., Prasad R. Treatment of     inflammatory bowel disease in a rodent model with the intestinal     growth factor glucagon-like peptide-2. J. Ped. Surg. 2000; 35:847-51 -   30. Cameron H., Yang P.-C., Perdue M. H. Glucagon-like     Peptide-2-enhanced Barrier Function Reduces Pathophysiology in a     Model of Food Allergy. Am. J. Physiol. 2003; 284: G905-G912 -   31. Ramsanahie A. P., Perez A., Duensing A. U., Zinner M. J.,     Ashley S. W., Wang E. E. Glucagon-like peptide 2 enhances intestinal     epithelial restitution. J Surg Res 2002; 107: 44-9 

1. A method for determining whether a compound potentially modulates glucagon-like peptide-2 (GLP-2) receptor response to a GLP-2 receptor ligand, comprising: incubating a first ex vivo intestinal tissue segment with the GLP-2 receptor ligand; measuring contractility of the first intestinal tissue segment; incubating a second ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound; and measuring contractility of the second intestinal tissue segment; wherein a difference between contractility of the first and second intestinal tissue segments is indicative of the compound potentially modulating GLP-2 receptor response to the GLP-2 receptor ligand.
 2. A method according to claim 1, wherein the first and second intestinal tissue segments are of mammalian origin.
 3. A method according to claim 2, wherein the first and second intestinal tissue segments are of human origin.
 4. A method according to claim 2 wherein the first and second intestinal tissue segments are of rat origin.
 5. A method according to claim 1, wherein the first and second intestinal tissue segments are small intestine tissue segments.
 6. A method according to claim 1 wherein the first and second intestinal tissue segments are colon tissue segments.
 7. A method according to claim 1, wherein the step of incubating the second ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound comprises the following ordered steps: adding the GLP-2 receptor ligand; and adding the compound.
 8. A method according to claim 1, wherein the step of incubating the second ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound comprises adding the GLP-2 receptor ligand substantially simultaneously with the addition of the compound.
 9. The method according to claim 1, wherein the GLP-2 receptor ligand is GLP-2 or a GLP-2 analogue.
 10. The method according to claim 9, wherein the GLP-2 receptor ligand is [gly2]GLP-2.
 11. A method for determining whether a compound potentially modulates glucagon-like peptide-2 (GLP-2) receptor response to a GLP-2 receptor ligand, comprising: incubating an ex vivo intestinal tissue segment with the GLP-2 receptor ligand; taking a first measurement of contractility of the intestinal tissue segment; incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound; and taking a second measurement of contractility of the intestinal tissue segment; wherein a difference between the first measurement of contractility and the second measurement of contractility is indicative of the compound potentially modulating GLP-2 receptor response to the GLP-2 receptor ligand.
 12. A method according to claim 11, comprising the following ordered steps: incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand; taking the first measurement of contractility of the intestinal tissue segment; washing the intestinal tissue segment; incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound; and taking the second measurement of contractility of the intestinal tissue segment.
 13. A method according to claim 11, comprising the following ordered steps: incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound; taking the second measurement of contractility of the intestinal tissue segment; washing the intestinal tissue segment; incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand; and taking the first measurement of contractility of the intestinal tissue segment.
 14. A method according to claim 11, wherein the intestinal tissue segment is of mammalian origin.
 15. A method according to claim 12, wherein the intestinal tissue segment is of human origin.
 16. A method according to claim 12 wherein the intestinal tissue segment is of rat origin.
 17. A method according to claim 11, wherein the intestinal tissue segment is a small intestine tissue segment.
 18. A method according to claim 11, wherein the intestinal tissue segment is a colon tissue segment.
 19. A method according to claim 11, wherein the step of incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound comprises the following ordered steps: adding the GLP-2 receptor ligand; and adding the compound.
 20. A method according to claim 11, wherein the step of incubating the ex vivo intestinal tissue segment with the GLP-2 receptor ligand and the compound comprises adding the GLP-2 receptor ligand substantially simultaneously with the addition of the compound.
 21. The method according to claim 11, wherein the GLP-2 receptor ligand is GLP-2 or a GLP-2 analogue.
 22. The method according to claim 21, wherein the GLP-2 receptor ligand is [gly2]GLP-2. 